Quaglia Daniela, Ebert Maximilian C C J C, Mugford Paul F, Pelletier Joelle N
Département de Chimie, Université de Montréal, Montréal, QC, Canada.
Center for Green Chemistry and Catalysis (CGCC), Université de Montréal, Montréal, QC, Canada.
PLoS One. 2017 Feb 8;12(2):e0171741. doi: 10.1371/journal.pone.0171741. eCollection 2017.
The Golden Gate strategy entails the use of type IIS restriction enzymes, which cut outside of their recognition sequence. It enables unrestricted design of unique DNA fragments that can be readily and seamlessly recombined. Successfully employed in other synthetic biology applications, we demonstrate its advantageous use to engineer a biocatalyst. Hot-spots for mutations were individuated in three distinct regions of Candida antarctica lipase A (Cal-A), the biocatalyst chosen as a target to demonstrate the versatility of this recombination method. The three corresponding gene segments were subjected to the most appropriate method of mutagenesis (targeted or random). Their straightforward reassembly allowed combining products of different mutagenesis methods in a single round for rapid production of a series of diverse libraries, thus facilitating directed evolution. Screening to improve discrimination of short-chain versus long-chain fatty acid substrates was aided by development of a general, automated method for visual discrimination of the hydrolysis of varied substrates by whole cells.
金门策略需要使用IIS型限制性内切酶，这类酶在其识别序列之外进行切割。它能够对独特的DNA片段进行无限制设计，这些片段能够轻松且无缝地重组。该策略已成功应用于其他合成生物学应用中，我们展示了其在工程化生物催化剂方面的优势应用。在南极假丝酵母脂肪酶A（Cal-A）的三个不同区域确定了突变热点，Cal-A作为生物催化剂被选作目标以证明这种重组方法的多功能性。对三个相应的基因片段采用了最合适的诱变方法（靶向诱变或随机诱变）。它们的直接重新组装使得能够在一轮操作中组合不同诱变方法的产物，从而快速产生一系列多样的文库，进而促进定向进化。通过开发一种通用的自动化方法来视觉区分全细胞对不同底物的水解作用，有助于筛选以提高对短链和长链脂肪酸底物的区分能力。
